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Transparent, nanostructured silk fibroin hydrogels with tunable mechanical properties Alexander Mitropoulos, Benedetto Marelli, Chiara Ghezzi, Matthew B Applegate, Benjamin P Paltrow, David L Kaplan, and Fiorenzo G Omenetto ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.5b00215 • Publication Date (Web): 26 Aug 2015 Downloaded from http://pubs.acs.org on September 8, 2015

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ACS Biomaterials Science & Engineering

Transparent, nanostructured silk fibroin hydrogels with tunable mechanical properties Alexander N. Mitropoulos1†, Benedetto Marelli1†, Chiara E. Ghezzi1, Matthew B. Applegate1, Benjamin P. Partlow1, David L. Kaplan1, and Fiorenzo G. Omenetto1,2* 1.

Department of Biomedical Engineering, Tufts University, 4 Colby St., Medford, MA,

02155, USA 2. Department of Physics, Tufts University, 4 Colby St, Medford, MA, 02155 USA *

Corresponding author: [email protected]

KEYWORDS silk, fibroin, hydrogel, transparency, cornea

Silk fibroin from the Bombyx mori caterpillar has been processed into many material forms, with potential applications in areas ranging from optoelectronics to tissue engineering. As a hydrogel, silk fibroin has been engineered as a substrate for the regeneration of soft tissues where hydration and mechanical compatibility are necessary. Current fabrication of silk fibroin hydrogels produces microstructured materials that lack transparency and limits the ability to fully exploit the hydrogel form. Transparency is the main characteristic of some human tissues (e.g. cornea) where silk fibroin in the film 1 ACS Paragon Plus Environment


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format has shown potential as scaffolding material, however, lacking the necessary hydration and successful attachment of cells without biochemical functionalization. Additionally, detection using light is an important method to translate information for instruction, sensing, and visualization of biological entities and light sensitive molecules. Here, we introduce a new method for the fabrication of transparent silk hydrogels by driving the formation of nanostructures in the silk fibroin material. These nanostructures are formed by exposing silk solution (concentration below 15 mg/ml) to organic solvents that induce the amorphous to crystalline transition of the protein and indeed the sol-gel transition of the material. We have also explored a novel process to modulate the mechanical properties of silk fibroin hydrogel within the physiological range by controlling the amount of metal ions present in the protein structure. Nanostructured silk fibroin hydrogels are biocompatible and allow for attachment and proliferation of human dermal fibroblasts without any biochemical functionalization. In addition, seeding of human cornea epithelial cells (HCECs) on the hydrogel surface results in the formation of an epithelium, which does not alter the gels’ transparency and shows biological properties that challenge the performances of HCECs seeded in collagen hydrogels, the current standard material for the engineering of corneal tissue.

Introduction The reinvention of silk fibroin as a sustainable material for biomedical, optics, photonics and electronics applications has been predicated on the numerous material formats (e.g. fibers, foams, particles, films, hydrogels) in which the protein can be

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processed after regeneration in aqueous solution.1 Additionally, silk fibroin materials can be engineered with tunable morphological, physical, mechanical and biological properties by fine regulation of molecular weight at the point of protein extraction from silk fibers during the removal of sericin and by controlling the protein conformation through exposure to heat, water vapor or polar solvents.1,2 For optics and photonics applications, the film format for silk has generated interest due to transparency, robust mechanical properties and preservation of heat-labile sensing molecules encapsulated within the protein, allowing for the fabrication of optic and photonic devices with unprecedented features that can be interfaced with biology.3 Conversely, other commonly used material formats of silk fibroin, such as foams and hydrogels are characterized by high optical loss due to internal light scattering.4–6 Silk hydrogels have been proposed as substrates for the engineering, modeling and regeneration of soft tissues, ranging from nerves to cartilage.7,8 There is in fact a need for soft biomaterials that match the physical and mechanical properties of human tissues by mimicking the hydrated nature of the extracellular space.9–11 In addition, silk hydrogels can be easily modified to provide appropriate morphological, biochemical and mechanical cues and can be functionalized with stabilized heat-labile compounds.12 Silk fibroin sol-gel transition occurs through inter- and intra-molecular interactions (mainly formation of hydrogen bonds and hydrophobic interactions) among protein chains, which fold from amorphous to thermodynamically stable beta-sheets, driven by exposure of silk solutions to shear forces, electric fields, pH near or below the isoelectric point (pI=3.83.9), polar solvents, heat and water removal.4,5 The micelle assembly process is also regulated by the strong amphiphilic (hydrophobic and hydrophilic domains) nature of the



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protein. Short hydrophilic spacers highly variable in their amino acid composition intervene between large hydrophobic blocks, where a sequence of six amino acids (GAGAGS) is continuously repeated. This recurrent motif plays a critical role in preventing premature -sheet formation and in modulating water solubility.13 Development of inter-molecular bonds results in aggregation of silk fibroin micelles into interconnected micron-sized particles with progressive loss of transparency of the silk solution, ultimately becoming a white hydrogel due to light scattering. Despite numerous applications of silk fibroin hydrogels in biomedical engineering, the lack of transparency has been of hindrance to fully capitalize on this material format.14 For example, biological entities (e.g. cells), light sensitive molecules (e.g. fluorescent, bioluminescent, photoactive macromolecules) and optogenetic tools can be incorporated into hydrogels for sensing and diagnostic applications, to generate biomimetic biological systems or to build optical interfaces with living tissues. Transparent hydrogels have been obtained with globular proteins by changing the molecular conformation to linear networks by heat denaturation but this is an unsuccessful approach with silk fibroin.4 Transparency is also the main characteristic of cornea, a tissue where silk fibroin has shown potential as scaffolding material for cornea replacements.15–22 Indeed, the possibility of combining optical clarity in the visible spectrum with the well-established, tunable biophysical, biochemical and biological properties of silk fibroin hydrogels would shine a new light on this material format, enabling the engineering of highly tunable tissue-equivalent constructs with enhanced optical and photonic functionalities. In this study we report a new gelation method to induce the formation of a silk fibroin gel where the protein vesicles present in solution aggregate into nanosized particles 4 ACS Paragon Plus Environment

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during the sol-gel transition of the material, forming a transparent gel with defined shape and dimensions that are maintained upon removal from a mold. In addition, the method allows for the fabrication of hydrogels with convex or concave geometries that enable the formation of optical components such as a lens.

Experimental section Silk fibroin solution preparation Silk fibroin solution was prepared as previously described (Figure 1a). B. mori silkworm cocoons were boiled for 30 minutes in a solution of 0.02 M Na2CO3 to remove the sericin glycoproteins. The extracted fibroin was rinsed in deionized water and set to dry for 12 h. The dried fibroin was dissolved in 9.3 M LiBr solution at 60°C for 3 h. The solution was dialyzed against deionized water using a dialysis cassette (Slide-a-Lyzer, Pierce, MWCO 3.5KDa) at room temperature for 2 days until the solution reached a concentration of ~60 mg/ml. The obtained solution was purified to remove large aggregates using centrifugation and filtered through a 5 µm syringe filter.

Hydrogel preparation Acetone Optima (Fisher Scientific) was used to synthesize the hydrogels. Acetone was set in a glass Petri dish and silk solution with a concentration of 10 mg/ml was added to an acetone bath (Figure 1b). The ratio of silk solution to acetone was of 2:1. The acetone was evaporated at room temperature while adding deionized water to exchange with the 5 ACS Paragon Plus Environment


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gels and to prevent the collapse of the gels. To improve the mechanical properties of the silk

fibroin

materials,

hydrogels

were

soaked

in

a

20

mM

solution

of

ethylenediaminetetraacetic acid (EDTA) (pH=8.5 with Tris base, Sigma Aldrich) for different time lengths from 1 h, 2 h, 7 h, 19 h, and 24 h. Hydrogels were then carefully rinsed in deionized water to remove any excess of EDTA. Type I collagen hydrogels (FirstLink UK, 2.1 mg/ml) were prepared according to the guidelines of the vendor protocol (final collagen fibrillar density of 0.2 wt%) and were used as control for cell culture.

Morphological characterization Scanning electron microscopy (SEM) was used to evaluate scaffold morphology. All the micrographs were taken with a Supra55VP FESEM (Zeiss) using the in-lens SE detector. Morphological characterization of the hydrogels was obtained by drying the samples in hexamethyldisilazane (HMDS). Samples were first dehydrated in a series of ethanol rinses at concentrations of 50%, 70%, 80%, 90%, 95%, 100%, and 100% for 30 minutes and then exposed to a series of HMDS baths at 70%, 90%, 100%, and 100% for 30 minutes to ensure complete saturation in HMDS. Samples were then left to dry in a chemical hood for 12 hours to allow complete evaporation of HMDS and then immediately sputter coated and imaged at 3kV.

Physical characterization


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For light transmission measurements, visible spectra were taken using a vis/near-infrared fiber-optic spectrometer (USB-2000, Ocean Optics). White light was propagated through the fiber to pass through the sample, and the transmitted light was coupled into a fiber tip guided to the spectrometer. The distance between the illumination source and the collection tip was fixed at 10 mm. All samples had a thickness of 4 mm. Dynamic light scattering (DLS) experiments were conducted using a Brookhaven Instrument BI200-SM goniometer (Holtsville, NY, USA) equipped with a diode laser operated at a wavelength of 532 nm. DLS analysis of the silk nanoparticles were mixed in glass vials at concentrations of 10 mg/ml, 20 mg/ml, 40 mg/ml, and 60 mg/ml and analyzed before gelation. Quantitative analysis of the distribution of relaxation times and corresponding size distributions were obtained using the Non-Negative Least Squares: Multiple Pass (NNLS) method. The size distribution extrapolated by the DLS was set to a weighted average to calculate the average diameter.

Raman microscopy measurements Raman spectra were collected using a JASCO NRS 3100 microRaman spectrophotometer (JASCO, Tokyo, Japan). Hydrogels were mounted on a glass microscope slide and excited at 784 nm with a laser focused using a 100x objective. Spectra were obtained in the 1800-200 cm-1 range using a resolution of 0.5 cm-1, 5 accumulations per sample and an exposure time of 20 s.



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Mechanical characterization Compressive properties of silk fibroin hydrogel were measured using an Instron 3366 testing frame (Instron, Norwood, MA) equipped with a 10 N load cell. Three different crosshead speeds (i.e. 0.100 mm/min, 0.200 mm/min, and 2.000 mm/min) were used to characterize the compressive properties of the hydrogels. Mechanical tests were conducted on hydrated samples at 22°C, 40% RH. The compressive modulus was calculated in the linear portion of the stress-strain curve.

Cell culture Human dermal fibroblasts (HDFa, Invitrogen) were seeded on silk hydrogels after treatment in ethanol for 24 h. Samples were rinsed in 3 subsequent PBS baths (pH=7.4, T=37°C) before cells were seeded at a density of 20,000 cells/cm2. Cells were cultured to confluence in high glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with GlutaMAX™ Supplement (Invitrogen), 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin antibiotic (Invitrogen). Cell cultures were maintained at 37°C in humidified atmosphere of 5% CO2. Human corneal epithelial cells (HCEC), isolated from the progenitor-rich limbal region of the eye, were purchased from Invitrogen. HCECs were cultured in Keratinocyte Serum Free Medium (Invitrogen) supplemented with 1% penicillin/streptomycin antibiotic at 37°C in humidified atmosphere of 5% CO2. HCECs at passage 3 were detached from tissue culture plastic using TrypLE™ Express (Invitrogen) and then reseeded on silk hydrogels and collagen hydrogels at a density of 25,000 cells/cm2. Cultures were 8 ACS Paragon Plus Environment

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incubated at 37°C for 3 h allowing the cells to attach to the hydrogel surface before the addition of culture media.

Metabolic activity AlamarBlueTM reagent was used to assess HCECs metabolic activity on silk fibroin hydrogel surfaces at days 1, 3, 7, and 10 in culture. Type I collagen hydrogels were used as control. For metabolic activity, samples were incubated in complete culture medium with 10% AlamarBlueTM reagent (Invitrogen, USA) at 37°C for 4 h. Post incubation, 100 µl aliquots of media were collected in triplicate from quadruple samples and the fluorescence detection, indicative of cellular reduction of resazurin indicator, was measured at 590 nm using 530 excitation using a microplate reader (SpectraMax M2, Molecular Devices, Sunnyvale, CA, USA). Acellular scaffolds were used as the background reference.

Cell imaging Confocal images were taken with a Leica DMIRE2 confocal laser-scanning microscope (Wetzlar, Germany). Live/dead sample staining was conducted using a LIVE/DEAD® Cell Viability/Cytotoxicity Kit (Life Technologies, Grand Island, NY, USA) by incubating a solution containing 2 µM calcein AM and 4 µM Ethidium homodimer-1 (EthD-1) for 60 minutes at 37°C. Samples were excited at 488 nm and emission at 510-



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530 nm for live cells (green) and excitation at 543 nm and emission for dead cells at 610640 nm (red). For SEM analysis, cell-seeded hydrogels were fixed in 10% buffered formalin for 12 h at 4°C. Samples were then washed in PBS (pH 7.4) three times before been dehydrated in a series of ethanol solutions at 50%, 70%, 80%, 90%, 95%, 100%, and 100% for 30 minutes. Samples were then critically point dried using an Auto Samdri 815 Series A (Tousimis, Rockville, MD) operating above the critical point of liquid carbon dioxide. All samples were sputter coated using platinum/palladium and imaged at 3 kV.

Statistical analysis All data were statistically compared with one way ANOVA tests using one way Anova tool (significance level = 0.05) using Origin Pro v.8 Software (OriginLab, USA).

Results and discussion Physical characterization of hydrogels A fast and robust method was developed to form transparent, nanostructured silk hydrogels by mixing two parts of silk fibroin solution (average molecular weight of 100kDa, 10 mg/ml) with one part of acetone (Figure 1, 2a and S1). Parameters such as silk fibroin concentration, average molecular weight, and choice of organic solvent were the determining factors in the formation and transparency of the hydrogels (Figure S2, S3 and S4). Silk fibroin concentration dictated the dimension of the silk particles within the 10 ACS Paragon Plus Environment

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forming hydrogel, and only silk solutions ≤ 15 mg/ml allowed to control silk fibroin particle diameter under 200 nm, resulting in optically clear hydrogels (Figure 2b, S3). Dynamic light scattering (DLS) measurements showed that mixing silk fibroin solution at concentrations ranging from 10 to 15 mg/ml with various polar organic solvents (i.e. acetone, ethanol, methanol, isopropanol) drives the assembly of silk micelles into submicron-sized particles (20 mg/ml) to alcohols and acetone (Figure S4). These data are also consistent with a previously reported study, where silk fibroin precipitation in an acetone reservoir allowed for the formation of uniform silk nanoparticles (98 nm diameter, polydispersity index 0.109), which were used as a controlled drug release system for chemotherapeutics.23 By modulating silk fibroin molecular weight (MW) at the point of sericin removal from the raw fiber, silk fibroin solutions with an average MW of 120 kDa (corresponding to 30 minutes boiling time24) provided the best trade-off between gel formation and transparency (Figure S2). Methanol, ethanol, isopropanol, and acetone have been used to induce amorphous to crystalline conformational change in silk fibroin, and mixing them with silk fibroin solution induce the formation of hydrogels.25 Here, gelation in acetone provided enhanced transparency when compared to the other organic solvents (Figure S2). In addition, acetone was successfully removed after the sol-gel transition occurred, as assayed with salicylaldehyde (see Supporting information). The nanomorphology of transparent silk hydrogels was also evident by scanning electron microscopy, which depicted the assembly of silk fibroin in materials with 11 ACS Paragon Plus Environment


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morphological features less than 100 nm (Figure 2c). The significance of material nanotopography has been previously demonstrated for stem cell differentiation26, cell adhesion27 and metabolic activity28 and the fabrication of nanostructured silk fibroin hydrogels through this gelation may be useful to probe biological activity.

Conditioning in Tris/EDTA Raman measurements corroborated the amorphous to higher ordered betasheet conformational change of silk fibroin during the sol-gel transition, as the Amide I and III scattering peaks of the protein shifted upon gelation from wavenumbers attributed to the amorphous silk (1661 cm-1 for Amide I, 1251 and 1276 cm-1 for Amide III) to -sheet features (1669 and 1230 cm-1 for Amide I and III, respectively) (Figure 2d).29,30 The control of higher order beta-sheet domains, or crystallinity, enables the modulation of the protein biodegradation in vivo and in vitro from hours to months and years.31–35 Enhanced crystallinity corresponds to a more packed, hydrophobic, structure that decreases accessibility by metalloproteinases (e.g. MMP1, MMP3, MMP9, MMP 13) and other proteolytic enzymes (e.g. chymotrypsin, trypsin) to cleavage sites in the protein.36 Concurrently, the modulation of crystallinity allows for the regulation of mechanical properties of the material as the expulsion of water from the protein structure together with the formation of inter-molecular hydrogen bonds result in enhanced elastic modulus and yield strength.37 To exploit the unique regulation of silk fibroin-based materials properties with hydrogels, it is common to modulate -sheet formation by exposing silk fibroin hydrogels to alcohols (e.g. ethanol and methanol).6 However, this


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type of treatment did not result in any significant impact effects on the conformation of the protein or on the mechanics of the hydrogels fabricated (data not shown), as the dehydration of the protein that drives the amorphous to crystalline transition was already achieved during the initial exposure to acetone during the sol-gel transition. We then pursued an unprecedented methodology to modify the properties of fibroin molecules upon gel formation, which was predicated on the removal of metal ions inherently present in the silk fibroin structure.38,39 Controlling the interplay between metal ions and proteins is in fact a versatile strategy to modulate the mechanical properties of structural proteins.38,40–44 By exposing silk fibroin hydrogels to solutions of aminopolycarboxylic acid, such as Tris/EDTA ([EDTA] =20 mM, T= 22°C, conditioning time up to 24 hours), we then explored the effects of chelating the ions involved in metal complexations with the fibroin protein. EDTA has in fact a strong tendency to form stable complexes with the divalent metal ions (Mg2+, Ca2+, Cu2+, and Zn2+) present in the secretory pathway of silk fibroin in B. mori.38 ICP-AES was used to measure the metal ion concentration in the hydrogels at increasing conditioning times in Tris/EDTA solution. There was an observed decrease in concentration of Mg2+, Ca2+, and Cu2+ ions in the samples conditioned in EDTA when compared to the not conditioned one (Figure S5a-d). Particularly, the Ca2+ concentration was reduced by almost 10 times compared to the starting concentration in the original hydrogels while Mg2+ and Cu2+ ions had a non-detectable concentration after conditioning for 24 h in EDTA. Interestingly, conditioning in Tris/EDTA solution had no apparent effect on the conformation of silk fibroin hydrogel, as there was no evident change in the FTIR spectra of the protein before and after the treatment (Figure S5e).



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Mechanical characterization of hydrogels Compressive tests were carried out to evaluate the effect of the Tris/EDTA treatment on the mechanical properties of the hydrogels. Figure 3 illustrates representative stress-strain curves at different crosshead speeds for increasing conditioning times in Tris/EDTA solution. All samples showed the densification behavior typical of soft material, where low compressive stress generates high material deformation. The typical viscoelastic behavior of silk hydrogels was also depicted, as increased crosshead speeds corresponded to increased stiffness. Silk fibroin hydrogels conditioned in Tris/EDTA solution showed a time-dependent enhancement of the mechanical properties; both compressive strength (Figure 3a) and compressive modulus (Figure 3b) increased. The time-dependent enhancement of mechanical strength was associated only with EDTA, as there was no statistically significant (p>0.05) change in mechanical properties of the hydrogels conditioned up to 24 hours in Tris solution only (Figure S6). In particular, by controlling the exposure time to EDTA it was possible to regulate the compressive modulus of the hydrogels within a range spanning two orders of magnitude (from 0.4±0.183 to 11.5±2.6 kPa), which correspond to the stiffness of many body tissues ranging from the brain to the muscle.45–47 Indeed, treatment of silk fibroin hydrogels in EDTA solution allows for the modulation their mechanical properties, which impacts cell differentiation and overall cell behavior.45–47

Biological characterization of hydrogels


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As a preliminary evaluation of biocompatibility, human dermal fibroblasts were cultured up to 28 days on silk fibroin hydrogels obtained with this gelation and treated with EDTA for 24 hours. Confocal laser scanning microscopy images were taken of the dermal fibroblasts at day 7, 14 and 28 after staining with calcein-AM fluorescein and EtBr-1 deoxyribonucleic acid binding (live/dead® assay) (Figures S7 and S8). Live/dead assay revealed the presence of HDFa at different depths within the hydrogel at day 7 in culture (Figure S7). At day 7, cells were viable (green) and attached to the surface of the hydrogel with negligible appearance of dead cells (red) (Figure S8a). In addition, a depth scan of the hydrogel revealed the presence of cells as deep as 1000 µm from the surface, showing that the human dermal fibroblasts penetrated the hydrogels and remained viable (Figure S7). SEM analysis of the cell-seeded hydrogels at day 7 showed deposition of extracellular matrix with a nanofibrillar structure, which can be associated to the formation of type I collagen from a fibroblastic cell-line. Prolonged culture times (days 14 and 28) of human fibroblasts on the silk fibroin hydrogels showed cell alignment and increased production of fibrillar nanostructures in the extracellular space (Figure S7 and S8). Optically clear hydrogels can find use in regenerative medicine for transparent tissue scaffolds. As a proof of principle, silk fibroin hydrogels were seeded with human cornea epithelial cells (HCECs) to evaluate potential use as cornea-equivalent materials for epithelium regeneration. Type I collagen was chosen as positive control material, due to the material track record in human clinical phase (Figure 4).48–54 A combination of confocal laser microscopy and live/dead® assay on HCEC-seeded silk fibroin hydrogels showed cell viability and proliferation over 10 days, with no appreciable difference when



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compared to collagen hydrogels. HCECs cultured on silk fibroin hydrogels also formed a visible epithelium at day 7 (Figure S9). In addition, metabolic activity of HCECs seeded on silk fibroin and collagen hydrogels was measured as a function of culture time at days 1, 3, 7, and 10 using reduction of AlamarBlueTM. The HCECs cultured on the silk fibroin hydrogels showed a similar (days 1, 3, 10, p>0.05) or increased (day 7, p

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